Our long term objectives are to identify gene mutations that cause obesity in humans and to understand their pathogenic effects. We have previously demonstrated that mutations in the Melanocortin-4 receptor (MC4-R), a hypothalamus expressed gene implicated in the central regulation of food intake, cause a common form of obesity in humans. This research proposal focuses on the pro-opiomelanocortin (POMC) gene. This gene is expressed in the arcuate nucleus of the hypothalamus and encodes the physiologic agonist ligands of the MC4R. Homozygous null mutations in the POMC gene result in a rare form of obesity associated with adrenal insufficiency and alterations in hair pigmentation. Recent results in the literature as well as our preliminary findings suggest that heterozygous POMC mutations can predispose to common obesity. Our hypothesis is that a significant number of common severe obesity cases are due to such heterozygous mutations in the POMC gene. To investigate this hypothesis we propose the following specific aims: Aim 1) To determine the prevalence and association of rare POMC gene variants in severe and or early onset human obesity. We will evaluate the prevalence of rare POMC mutations in severe obesity by systematically screening cohorts of severely obese children and adults for mutations in the POMC coding sequence. We will verify the absence of such mutations in non-obese controls. We will analyze the segregation of obesity with the mutations in the families of the probands. We will search for specific phenotypes distinguishing obese POMC mutation carriers from other obese patients. Aim 2) To determine the in vitro effects of obesity associated POMC mutations. Starting with the known obesity-associated heterozygous mutations in the POMC gene, we will systematically evaluate the effects of all the obesity-associated POMC mutations. Specifically we will: 2.1- Examine the effects of human obesity associated POMC mutations on the processing of POMC. 2.2- Examine the effects of mutated human obesity associated POMC derived peptides on MC4R and MC3R activation. 2.3- Test for a toxic effect of human obesity associated POMC mutations on primary cultures of neurons. Aim 3) To determine the effects of obesity associated POMC mutations in vivo. To further confirm the pathogenicity of human obesity associated POMC mutations we will: 3.1- Evaluate the short term effects of mutated human obesity associated POMC derived peptides on food intake in rats. 3.2- Evaluate the long-term effects of transgenic over-expression of human obesity associated POMC mutations in mice. This work will be an additional step towards the genetic classification of obesity in humans and will contribute to the development of targeted preventive and therapeutic interventions based on the underlying genetic defects in the patients. [unreadable] [unreadable]